A murine monoclonal antibody, MLS128, that was assigned to an anti-Tn antibody has been established by immunizing with human colonic cancer cells (LS180cell). MLS128 bound to mucin glycopeptides from LS180 cells and their asialo forms to the same extent as well as to submaxillary mucin (OSM) and asialo OSM.To determine the epitopic structure for an anti-Tn monoclonal antibody, MLS128, asialo OSM was digested with various proteases, and the digests were fractionated by immunoaffinity chromatography and high performance liquid chromatography. From the tryptic digest, a glycopeptide, GP-I, and five other glycopeptides, GP-1-5, were obtained as bound and unbound fractions, respectively, of the immunoaffinity column. By solid phase radioimmunoassay, it was found that GP-I was strongly immunoreactive. On treatment with V8 protease, GP-I was converted to two glycopeptides, one with poor reactivity and the other with intermediate reactivity.From the thermolysin digest, the smallest fragment, GP-II, was isolated, which was strongly immunoreactive as GP-I. GP-II corresponded to a part of GP-I, its sequence being Leu-Ser*-Glu-Ser*-Thr*-Thr*-Gln-Leu-Pro-Gly, where asterisks denote amino acids to which an GalNAc residue is attached. Other anti Tn monoclonal antibodies, NCC-LU-35 and CA3239, showed essentially the same reactivity to these glycopeptides as MLS128 did. The glycopeptides (GP-1-5), which exibited poor immunoreactivity, contained various GalNAc-containing structures, such as GalNAc-Thr/Ser, and GaINAc-Ser/Thr-(GalNAc)-Ser/Thr. These results indicate that a glycopeptide including a cluster structure, Ser*-Thr*-Thr*, is an essential part of the epitope recognized by anti-Tn antibodies. We also found that Tn antigen is expressed on leukosialin of T-lymphoid cell line, Jurkat and the Tn antigen is more complex than GalNAc-Ser/Thr.